U.S. patent application number 10/519541 was filed with the patent office on 2006-06-22 for chitosan/acidic biopolymer hybrid fiber and culture base for animal cells.
This patent application is currently assigned to Chemical Biology Institute. Invention is credited to Tadanao Funakoshi, Kazuo Harada, Norimasa Iwasaki, Nobuhiki Maekawa, Tokifumi Majima, Akio Minami, Shin-ichiro Nishimura, Sachiko Nonaka, Seiichi Tokura.
Application Number | 20060134158 10/519541 |
Document ID | / |
Family ID | 29996896 |
Filed Date | 2006-06-22 |
United States Patent
Application |
20060134158 |
Kind Code |
A1 |
Majima; Tokifumi ; et
al. |
June 22, 2006 |
Chitosan/acidic biopolymer hybrid fiber and culture base for animal
cells
Abstract
It is intended to provide a three-dimensional scaffolds
appropriate for culturing animal cells such as chondrocytes and
fibroblasts. These three-dimensional scaffolds which comprise a
chitosan/acidic biopolymers hybrid fibers composed of chitosan or
its salts in the inside of the fiber and complexes of chitosan with
biodegradable acidic biopolymers coating the fiber surface, are
made of fibers capable of retaining their shapes after allowing to
stand Dulbecco's modified Eagle's medium (DMEM) containing 10% of
FBS (fetal bovine serum) at room temperature for 2 weeks.
Inventors: |
Majima; Tokifumi;
(Sapporo-shi, JP) ; Iwasaki; Norimasa;
(Sapporo-shi, JP) ; Funakoshi; Tadanao;
(Sapporo-shi, JP) ; Minami; Akio; (Sapporo-shi,
JP) ; Nishimura; Shin-ichiro; (Sapporo-shi, JP)
; Tokura; Seiichi; (Suita-shi, JP) ; Harada;
Kazuo; (Sapporo-shi, JP) ; Nonaka; Sachiko;
(Sapporo-shi, JP) ; Maekawa; Nobuhiki;
(Sapporo-shi, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Chemical Biology Institute
2-1, Utsukushigaoka 4-jo, 9-chome Kiyota-ku
Sapporo-shi, Hokkaido
JP
004-814
|
Family ID: |
29996896 |
Appl. No.: |
10/519541 |
Filed: |
June 26, 2003 |
PCT Filed: |
June 26, 2003 |
PCT NO: |
PCT/JP03/08080 |
371 Date: |
December 28, 2004 |
Current U.S.
Class: |
424/422 ;
435/366; 442/128 |
Current CPC
Class: |
A61L 27/58 20130101;
A61L 27/48 20130101; C12N 5/0655 20130101; C12N 2533/72 20130101;
A61L 27/20 20130101; A61K 35/12 20130101; A61L 27/3817 20130101;
A61L 27/3895 20130101; D01F 8/16 20130101; C12N 2533/74 20130101;
A61L 27/48 20130101; Y10T 442/2566 20150401; A61L 27/20 20130101;
A61L 27/3843 20130101; A61L 27/3804 20130101; C12N 5/0068 20130101;
C08L 5/08 20130101; C08L 5/08 20130101 |
Class at
Publication: |
424/422 ;
435/366; 442/128 |
International
Class: |
C12N 5/08 20060101
C12N005/08; B32B 27/04 20060101 B32B027/04; A61F 13/00 20060101
A61F013/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 28, 2002 |
JP |
2002-190674 |
Claims
1. A chitosan/acidic biopolymers hybrid fibers in which the inner
part of the fibers comprises chitosan or salts thereof and the
surface of the fibers are covered by a complex of chitosan and a
biodegradable acidic biopolymers and which retains the form thereof
when the fibers are soaked in DMEM medium (Dulbecco's Modified
Eagle's Medium) at room temperature for 2 weeks.
2. A chitosan/acidic biopolymers hybrid fibers of claim 1 in which
the acidic biopolymers are selected from the group consisting of
hyarulonic acid, alginic acid, chondroitin sufate, dermatan
sulfate, heparin, heparan sulfate, keratan sulfate and polyglutamic
acid.
3. A method for preparing the fibers of claim 1 which comprise the
steps of: 1) dissolving chitosan in an aqueous acid solution to
prepare an aqueous solution of chitosan salt; 2) wet spinning the
aqueous solution of chitosan salt using alkaline earth metal salts
as coagulants to form fibers; 3) immersing the fibers in a solution
of biodegradable acidic biopolymers to react chitosan with acidic
biopolymers on the surface of the fibers to form chitosan/acidic
biopolymer hybrid fibers; 4) optionally stretching the hybrid
fibers; and 5) treating the fibers with bases, di- or more-basic
inorganic acids or salts thereof, tri- or more-basic organic acids
or salts thereof.
4. A method for preparing the fibers of claim 1 which comprise the
steps of: 1) dissolving chitosan in aqueous acid solutions to
prepare aqueous solutions of chitosan salt; 2) wet spinning the
aqueous solution of chitosan salt using bases, di- or more-basic
inorganic acids or salts thereof, tri- or more organic basic acids
or salts thereof as a coagulant to form fibers; 3) immersing the
fibers in a solution of biodegrabable acidic biopolymers to react
chitosan with the acidic biopolymers on the surface of the fibers
to form chitosan/acidic biopolymer hybrid fibers; and 4) optionally
stretching the hybrid fibers.
5. Three dimensional scaffolds for animal cells comprising the
fibers of claim 1.
6. Three dimensional scaffolds of claim 5 in which the animal cell
is chondrocyte.
7. Three dimensional scaffolds of claim 5 in which the animal cell
is fibroblast.
8. Three dimensional scaffolds of claim 5 in which the animal cells
are undifferentiated cells.
9. A method for culturing chondrocytes comprising culturing
chondrocytes cells in vitro using the three dimensional scaffolds
of claim 6.
10. A method for culturing chondrocytes of claim 9 in which a
growth factor is added during culturing.
11. A method for culturing of claim 9 or 10 in which the culturing
is effected under a low oxygen condition of 1 to 15% and/or under a
pressure of 0.1 to 20 MPa.
12. A method for culturing fibroblasts comprising culturing
fibroblasts in vitro using the three dimensional scaffolds of claim
7.
13. A method for culturing fibroblasts of claim 12 in which a
growth factor is added during culturing.
14. A method for culturing fibroblasts of claim 12 or 13 in which
culturing is effected with a stretch stimulus of 0.01 to 50 mm/cm
being added.
15. A method for culturing animal cells comprising culturing
undifferentiated cells in vitro using the three dimensional
scaffolds of claim 8.
Description
TECHNICAL FIELD
[0001] The present invention relates to chitosan/acidic biopolymer
hybrid fibers and the method for preparation of the same. The
present invention is also related to a three dimensional scaffolds
for animal cells comprising the fibers and a method for culturing
animal cells using the scaffolds.
BACKGROUND OF THE INVENTION
[0002] We are now entering senior citizen society and the number of
patients suffering from arthritis reach one percent of the
population. Most of the patients have osteoarthritis or chronic
rheumatism caused by damage or aneretic death of cartilage tissue.
Since cartilage regeneration ability is very low, artificial
prosthetic devices were implanted. However, there are many problems
in this method such as inflammation by elution of metal ion from
inserted material, looseness with bone joining portion and
durability. Since lifetime thereof are about ten years, artificial
prosthetic devices cannot be permanently curable treatment.
[0003] In the treatment of damage of ligament (in particular
anterior cruciate ligament which fixes knee joint) and tendon,
autologous transplantation of healthy host ligament or tendon is
done. However, muscle strength on healthy host region decreases to
about one half, resulting in the obstruction of exercise ability.
Also, the amount of donor tissue available for transplantation is
limited. Reconstructive surgery in which artificial ligament
consisting of synthetic fibers was done. However, since cells are
not adhered to the synthetic fibers, it was worn out with the lapse
of time and is not used now.
[0004] Accordingly, the tissue engineering for tissues or organs of
low self-repair potential are being vigorously studied in which
autologous cells at healthy region are isolated, cultured to
proliferate and differentiate, and then implanted again into same
patients to regenerate them. Since isolated cells proliferate and
differentiate by adhering on a surface of some substance, the
development of excellent artificial extracellular matrix (referred
to as "scaffold" hereinafter) is essential.
[0005] Generally, the conditions required for scaffolds are:
[0006] 1) that it is not inflammatory and biocompatible;
[0007] 2) that it is biodegradable;
[0008] 3) that it is excellent for cell adhesion;
[0009] 4) that it can maintain the cell activity; and
[0010] 5) that it has three dimensional structure which enable
tissues to regenerate by growth, migration and differentiation of
cells.
[0011] In addition, compressive stress up to about 20 MPa is
applied to articular cartilage, and high tensile stress is applied
to ligament and tendon. Accordingly, the scaffolds are further
required
[0012] 6) that it maintain shape stability after implantation;
and
[0013] 7) that it has mechanical strength.
[0014] From the view of tissue engineering, interstitial cells such
as fibroblast, smooth muscle cell and endothelial cell are buried
in a three dimensional framework (scaffolds) consisiting of
biodegradable materials such as polyglycolic acid, cotton,
cellulose, gelatin, collagen, polyhydroxyalkanoate, or
non-biodegradable materials such as polyaramid, polyester,
polystyren, polypropyren, polyacrylate, polyvinyl compound,
polycarbonate, polytetrafuolroethylene, nitrocellulose, and
cultured to prepare interstitial tissue which pack three
dimensional structure crosslinked by interstitial cells and
connective tissue protein which interstitial cells naturally
secrets (Japanese Patent Kohyo 50611/1999).
[0015] As scaffolds of fibroblast for ligament or tendon repair
with natural polymer, sponge or fiber of collagen (Dunn, M. G. et
al., J. Biomed. Mater. Res., 29:1363-1671 (1955)), sponge-like
structures in which glucosaminoglycan is bonded to collagen
(Torres, D. S., et al., Biomaterials 21:1607-1619 (2000)),
materials in which collagen is bonded to the surface of polylactic
acid fiber (Ide, A., et al., Material Sci. Eng., C17:95-99 (2001)),
or materials in which collagen is crosslinked with
nordihydroguaseletinic acid (Koob, T. J. wt al., J. Biomed. Mater.
Res., 56:40-48 (2001)) were used to examine the proliferation of
fibroblast and the regeneration of ligament tissue.
[0016] However, in the method in which collage is used, since
collagen scaffolds are allogenic, there is high probability that
antigenic property and infection associated with it become serious
problems. Furthermore, collagen gels and collagen materials do not
offer the same flexibility for modification that might be achieved
with biodegradable synthetic polymer. The lack of elasticity in
collagen-based materials is an important problem as scaffolds of
ligament or tendon.
[0017] Fiber or sponge in which polyglycolic acid or polylactic
acid is used as biodegradable scaffolds for chondrocytes have been
studied (Japanese Patent Kokoku No. 6155/1994; Japanese Patent
Kohyo No. 511679/1996; Ito, K. et al., Mat Res. Soc. Proc.,
252:359-365 (1992); Freed, L. E., et al., J. Biol. Mater. Res.,
27:11-23 (1993)). These materials have advantages that they have no
toxicity due to the identity of hydrolysis product thereof with
physiological metabolite, have mechanical strength due to large
polymer, and are easy to be molded. However, since these
biodegradable synthetic polymers have not adhesiveness to cell, an
attempt to increase cell adhesion by fixing biopolymer such as RGD
(arginine-glysin-aspartic acid tripeptide) which is a cell adhesive
factor in vivo, gelatin, and collagen on the surface of the
material has been made (Yamaoka, T. et al., J. Biol. Macromol.,
25:265-271 (1999)). However, such chemical fixing methods are
laborious in manipulation, and possible residence of the reagents
used for fixing reaction is concerned.
[0018] On the other hand, as a scaffold of chondrocyte using
biopolymer, collagen gel or sponge-like materials (Japanese Patent
Kokai No. 22744/1994; Japanese Patent Kohyo 510639/1999; Japanese
Patent Kokai No. 224678/2001; Fujisato, T., et al Biomaterial
21:153-159 (2000)) and sponge-like materials composed of chitin and
chitosan were examined (Park, Y. J., et al., Biomaterial 21:153-159
(2000)). However these materials have problem in shape stability
and mechanical strength. Collagen is expensive in material cost and
infection with BSE is concerned.
[0019] Scaffolds consisting of a complex of acidic (anionic) and
basic (cationic) polymers are reported (Japanese Patent kokai Nos.
335382/1994 and 277038/1994). The scaffold has a form of plate or
film structure prepared by mixing both solutions and drying, and
has not a three-dimensional porous structures composed of fibers.
There are also described the method in which the above solution is
coated or sprayed on an appropriate materials such as glass, metal
or plastic. However, since the scaffolds of the whole are not
biodegradable, they are not suitable as scaffolds for the
regeneration of cartilage tissue in which biodegradability is
required.
[0020] The method for preparing hybrid fiber consisting of
biodegradable acidic polymer and basic polymer is reported
(Japanese Patent kokai No 2002-291461). However, in the method for
preparing the fiber, alginic acid is used as main material and a
very small amount of basic polymer is added. The fiber is
hydrophilic, swelled in medium and thus has not shape stability.
The three dimensional scaffolds prepared by dissolving chitosan in
acetic acid, spinning in calcium chloride solution, immersing in
hyarulonic acid solution, reeling, drying, preparing fiber in a
form of sheet, applying chitosan around the sheets and piling up
the sheets are reported (Iwassaki et al., The 75th Japan Orthopedic
Society Research Meeting, May 16 to 19, 2002 (okayama); Yamane et
al., The 16th Japan Orthopedic Society Basic Research Meeting, Oct.
18, 2001 (Hiroshima); Yamane et al., The 20th Japan Implantation
and Regeneration Medicine Meeting, Oct. 27, 2001 (Kyoto)). However,
since the fibers of chitosan are made up as acetate salt, the fiber
produced is gradually swelled or dissolved in medium, resulting in
non-stability of shape and decreased growth of chondrocyte within
the fiber.
PROBLEM TO BE SOLVED BY THE INVENTION
[0021] It is an object of the present invention to provide
scaffolds for animal cell which satisfies all the requirements
above-mentioned. That is, it is an object of the present invention
to provide scaffolds for animal cell having the following
properties:
[0022] 1) In the culture of animal cells, the seeding of cells is
easy. Seeded or propagated animal cells can be easily adsorbed on
and adhered to the scaffolds.
[0023] 2) Animal cell such as chondrocyte and fibroblast propagate
on the surface or inside of the scaffolds and secret extracellular
matrix to form a connective tissue.
[0024] 3) The scaffolds of the present invention have three
dimensional space occupied by connective tissue formed.
[0025] 4) The scaffolds of the present invention has sufficient
mechanical strength till connective tissue is regenerated after
implantion
[0026] 5) The scaffolds of the present invention is biocompatible
and biodegradable, and then, ultimately disappears after the
regeneration of connective tissue.
SUMMARY OF THE INVENTION
[0027] The present invention relates to a chitosan/biodegradable
acidic biopolymer hybrid fibers in which the inner part of the
fibers comprises chitosan or salts thereof and the outer part of
the fibers is covered by a complex of chitosan and biodegradable
acidic biopolymers and which retains the form thereof when the
fibers are soaked in DMEM medium (Dulbecco's Modified Eagle's
Medium) supplemented with 10% FBS (fetal bovine serum) at room
temperature for 2 weeks. The term "retain form" means has the
original form without dissolving or swelling.
[0028] The present invention is also related to a method for
preparing the fibers which comprise the steps of:
[0029] 1) dissolving chitosan in an aqueous acid solution to
prepare an aqueous solution of chitosan salt;
[0030] 2) wet spinning the aqueous solution of chitosan salt using
alkaline earth metal salts as coagulant to form fibers;
[0031] 3) immersing the fibers in solution of biodegradable acidic
biopolymers to react chitosan with acidic biopolymer on the surface
of the fibers to form chitosan/acidic biopolymer hybrid fibers;
[0032] 4) optionally stretching the hybrid fibers; and
[0033] 5) treating the fibers with a base, a di- or more-basic
inorganic acid or salt thereof, or a tri- or more-basic organic
acid or salt thereof.
[0034] The present invention relates to a method for preparing the
fibers which comprises the steps of:
[0035] 1) dissolving chitosan in an aqueous acid solution to
prepare an aqueous solution of chitosan salt;
[0036] 2) wet spinning the aqueous solution of chitosan salt using
a base, a di- or more-basic inorganic acid or salt thereof, or a
tri- or more-basic organic acid or salt thereof as a coagulant to
form fibers;
[0037] 3) immersing the fibers in a solution of biodegradable
acidic biopolymers to react chitosan with acidic biopolymers on the
surface of the fibers to form chitosan/acidic biopolymer hybrid
fibers; and
[0038] 4) optionally stretching the hybrid fibers.
[0039] The present invention further relates to a three dimensional
scaffolds for animal cells comprising the fibers. Animal cells
include, but not be limited to, chondrocyte, fibroblast, nervous
cell and undifferentiated cells which are subsequently
differentiated into those cells.
[0040] The present invention relates to a method for culturing
animal cells comprising culturing animal cells in vitro using the
three dimensional scaffolds.
[0041] The present invention relates to a scaffold for grafts which
are obtained by the above culture and in which proliferated animal
cells are bonded to the scaffolds.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 is a reproduction of an optical micrograph of
chondrocyte after 21 days of culture using the three dimensional
scaffolds of the present invention
[0043] FIG. 2 is a reproduction showing the result of Alcian Blue
Safranine staining of chondrocyte after 21 days of culture
[0044] FIG. 3 is a graph showing the amount of protein and acidic
mucopolysaccharide produced by chondrocyte per the three
dimensional scaffold after 1, 7, 14 and 21 days of culture.
[0045] FIG. 4 is a reproduction of optical micrograph of tissue
stained with Safranin O. A defective portion on articular cartilage
tissue of rabbit knee was prepared. To this place, rabbit
chondrocytes cultured on the three dimensional scaffolds were
implanted. On 8 weeks after the implantation, the defective portion
was subjected to laparotomy and tissue observation was
effected.
[0046] FIG. 5 is a graph showing the amount of DNA of fibroblast
per the three dimensional scaffold after 1, 7, 14 and 21 days of
culture.
[0047] FIG. 6 is a reproduction of a optical micrograph showing the
result of type I collagen immunohistostaining by
streptavidin-biotin method using anti-mouse antibody.
[0048] FIG. 7 is a reproduction of a scanning electro microscope
image of fibroblasts cultured for 28 days on three dimensional
scaffolds.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0049] The inventors contrived two methods for preparing
chitosan/acidic biopolymer hybrid fibers in which the inner part of
the fibers comprises chitosan or salts thereof and the outer part
of the fibers is covered by a complex of chitosan and biodegradable
acidic biopolymers and which retains the form thereof when the
fibers are soaked in DMEM medium (Dulbecco's Modified Eagle's
Medium) supplemented with 10% FBS at room temperature for 2
weeks.
[0050] In the first method, chitosan which is a basic polymer is
dissolved in an aqueous acidic solution to prepare an aqueous
solution of chitosan salt. Acids to be used may be inorganic acids
or organic acids. Preferred examples of inorganic acids are
monobasic acids such as hydrogen chloride and nitric acid.
Preferred examples of organic acids are formic acid, acetic acid,
propionic acid, butyric acid, ascorbic acid, and the like.
[0051] The aqueous solution of chitosan salt is subjected to wet
spinning to form fibers. "Wet spinning" is a method in which
spinning solution of polymer is extruded through a nozzle into a
coagulating bath having high ability to desolvate to coagulate the
polymer.
[0052] Water soluble salt of alkaline earth metals such as calcium,
magnesium, barium, for example, halogen salts, can be used as a
coagulating agent. Calcium chloride is most preferred. The alkaline
earth metal salt is dissolved in water, or a mixture solvent of
water/methanol and used. The concentration of the alkaline earth
metal salt is from 10% to a saturated concentration, preferably
from 40% to 60%.
[0053] The chitosan salt solution is extruded through a nozzle into
a coagulating bath containing the coagulating agent to coagulate
chitosan to form fibers. The fibers formed are immersed into a
mixture solvent of water-miscible solvent and water to remove
excessive coagulant.
[0054] The term "acidic biopolymers" means polymers derived from
natural source having acidic groups such as carboxyl group, sulfate
group, sulfonate group, phosphate group, or salt thereof. In a
preferred embodiment, the biopolymers are polysaccharides. "Acidic
biopolymers" also include molecular weight decreased naturally
occurring biopolymer or acidic groups formed naturally occurring
biopolymer by any physical, chemical or enzymatic means.
[0055] Examples of the acidic biopolymers containing carboxyl
groups include polymers containing gluconic acid, glucuronic acid,
iduronic acid, D-mannuronic acid, galactruronic acid, gluronic
acid, sialic acid, and glutamic acid, for example, hyaluronic acid,
alginic acid, heparin, or polyglutamate.
[0056] Examples of the acidic biopolymers containing sulfate groups
include chondroitin sulfate, dermatan sulfate, heparin, heparan
sulfate, keratan sulfate. Examples of the acidic biopolymers
containing phosphate groups include DNA and RNA. Two or more kinds
of these acidic biopolymers may be used for the preparation of a
complex of chitosan and acidic biopolymers.
[0057] The complex of chitosan and acidic biopolymers means a
complex formed by electrostatic interaction between cationic group
of chitosan and anionic group of acidic biopolymers. Chitosan may
be contacted with acidic biopolymers in an appropriate medium, for
example, water, to form the complex. It was found that for the
formation of the complex, it is not necessary to combine both the
aqueous solutions. Solid chitosan may be contacted with acidic
biopolymer solutions.
[0058] Solution in which an acidic biopolymer is dissolved, for
example, in water or water/alcohol is prepared. The concentration
of acidic biopolymer is 0.01% to 10%, preferably 0.05% to 1%. When
the chitosan fibers above are immersed in the acidic biopolymer
solution, a complex of chitosan (basic polymer) and acidic
biopolymer is formed on the surface of chitosan fibers. The
resulting fibers are hybrid fibers in which the inner portion
comprises chitosan and the outer surface is coated with the complex
of chitosan (basic polymer) and acidic biopolymer. The fibers may
be optionally extruded. The formation of the complex may be
effected after extrusion.
[0059] The thus resulting fibers are after-treated with an aqueous
solution of inorganic base, di or more-basic inorganic acid or salt
thereof, or tri- or more-basic organic acid or salt thereof, or a
solution thereof using mixture solvent of water and water miscible
organic solvent. Without the after-treatment, the fibers may be
gradually dissolved in culturing medium and thus has not shape
stability as a scaffold. The examples of inorganic bases include
alkaline metal hydroxide such as NaOH and KOH. Examples of di- or
more-basic inorgan acids include sulfuric acids and phosphoric
acids. Examples of salts thereof include sodium carbonate, sodium
bicarbonate, trisodium phosphate, sodium dihydrogenphosphate,
disodium hydrogenphosphate, sodium sulfate, sodium hydrogensulfate
and potassium salts and ammonium salta corresponding to such sodium
salt. Examples of tri- or more-basic organic acids include citric
acid, ethylenediamine tetraacetic acid, 1,1,2-tricaboxyethane,
1,1,2-tricaboxy-2-methylethane, 1,1,3-tricaboxypropane,
1,2,3-tricaboxypropane, 1,1,2,2-tertracaboxyethane, and
1,2,2,3-trtracaboxyepropane. The concentration of these
after-treatment agents is 0.1 to 10%. It is preferably 0.2 to 1%
for alkaline metal hydroxides and 1 to 3% for inorganic acids and
organic acids. After the treatment, the fibers are thoroughly
washed with water and immersed in methanol to dehydrate and dry
them.
[0060] The second method for preparation of the fibers is different
from the first method for preparation of the fibers in that
inorganic base, di- or more-basic inorganic acids or salts thereof,
or tri- or more-basic organic acids or salts thereof is used as a
coagulant, and that the after-treatment is not effected. That is,
chitosan is dissolved in acidic aqueous solution to form an aqueous
solution of chitosan salt. The aqueous solution is subjected to wet
spinning using inorganic bases, di- or more-basic inorganic acids
or salts thereof, or tri- or more-basic organic acids or salts
thereof as a coagulant to form fibers. The resulting fibers are
then immersed in a mixture solvent of water-miscible organic
solvent and water to remove excessive coagulants. The fibers are
immersed in a solution of biodegradable acidic biopolymers to react
chitosan with acidic biopolymer on the surface of the fibers to
form chitosan/acidic biopolymer hybrid fibers. The fibers may be
optionally extruded. Examples of inorganic bases, di- or more-basic
inorganic acids or salts thereof, or tri- or more basic organic
acids or salts thereof as a coagulant are the same as mentioned as
the after-treatment agents in the first method for the hybrid
fibers preparation above. The concentration of the coagulant is in
the range of 0.5% to 30%. The concentration of chitosan, the kind
of acid necessary to disolve chitosan, the concentration of acidic
biopolymers may be the same as in the first method for
preparation.
[0061] The chitosan/acidic biopolymer hybrid fibers thus prepared
in which the inner portion of the fibers comprises chitosan and the
surface is covered with the complex of chitosan and acidic polymers
has considerable mechanical strength, can be adhered to animal cell
such as chondrocyte and fibroblast, and is biodegradable and
biocompatible.
[0062] In order to use as implanting scaffold for animal cells, the
scaffolds must be a three dimensional structure having a space in
which cell can proliferate and which retain extracellular matrixes
secreted from the cells. The three dimensional scaffolds must also
have form-stability and mechanical strength when strength is
applied to them. The three dimensional scaffolds which satisfy the
above requirement can be produced from the texture, knitting or
interlace prepared from the hybrid fibers prepared by the method of
the present invention. The texture, knitting or interlace can be
produced by conventional methods. The texture, knitting or
interlace may be folded or piled as necessary to give thickness.
The texture, knitting or interlace which is folded or piled is
unified using the hybrid fibers of the present invention. The
texture, knitting or interlace folded or piled may contain the
fiber in the inside in a form other than these forms. The form of
the three dimensional scaffolds may be changed in accordance with
injury portion.
[0063] The three dimensional scaffolds of the present invention
thus have the following advantageous properties as scaffolds.
[0064] 1) In the culture of animal cells such as chondrocyte and
fibroblast, seeding of cell is easy. Seeded or proliferated animal
cells such as chondrocyte and fibroblast can be adsorbed on or
adhered to scaffolds.
[0065] 2) Animal cells such as chondrocyte and fibroblast
proliferate on the surface or inside of the scaffolds and secret
extracellular matrixes to form a connective tissue.
[0066] 3) The scaffolds of the present invention have three
dimensional space which can be occupied by formed connective
tissue.
[0067] 4) The scaffolds of the present invention have sufficient
mechanical strength till connective tissue is regenerated after
implantation
[0068] 5) The scaffolds of the present invention are biocompatible
and biodegradable and ultimately disappears after the regeneration
of the tissue.
[0069] The culture of animal cells using the three dimensional
scaffolds of the present invention can be effected pursuant to
conventional method for culturing animal cell (see, for example,
Klagsburn, M., "Large Scale Preparation of Chondrocytes", Methods
in Enzymol., 58:560 (1979)). First of all, the scaffolds are
subjected to disinfection treatment by heating in an autoclave or
by gas in such a manner that the forms and properties of the
scaffolds are not disrupted. Animal cells are then seeded as evenly
as possible on the three dimensional scaffolds and cultured. Cells
used for culture may be derived from mammal such as rabbit, cattle,
horse, dog, cat, and human cell. Preferred are human cells. Most
preferred are cells from patient which undergos implantation.
[0070] Medium used in conventional animal cell culture, for
example, DMEM (Dulbecco's Modified Eagle's Medium) containing human
serum can be used. Any growth factor such as TGF-beta (transforming
growth factor-beta), FGF (fibroblast growth factor), ChM-1
(chondromojelin-1) may be added to the medium.
[0071] In order for seeded cells to proliferate and differentiate
on scaffolds, it is crucial to have high cell adhesion and
adsorption.
[0072] Since cartilage tissue is under low oxygen condition due to
lack of blood vessel in it and suffers pressure loading due to body
weight, the culture under the condition similar to these
physiological condition may be effective. Accordingly, it is
possible to effect the culture under low oxygen concentration of 1
to 15%, or under pressure of 0.1 to 20 MPa (0.01 to 2 Hz in the
case of cyclic applying), or under combined condition of these
conditions. Specifically, the method for applying pressure include
a method for applying air pressure or water pressure to the medium
using a pump or a piston.
[0073] Since stretching stress is applied to ligament and tendon,
it might be effective to culture under the condition similar to
physiological stretching conditions. Accordingly, in the culture of
fibroblast, culture may be effected under a stretching stimulus of
0.01 to 50 mm/cm (0.01 to 2 Hz in the case of cyclic applying).
Specifically, applying of stretching stress is effected by adding
stretch and contraction change while both ends of the scaffolds
immersed in medium are fixed to an instrument having an ability to
stretch and contract.
[0074] Culture of chondrocyte is at least continued till
extracellular matrixes are formed. Usually, by 2 to 4 weeks,
chondrocytes are adhered on the surface of three dimensional
scaffolds of the present invention, proliferate and secret
extracellular collagen-like matrix.
[0075] The three dimensional scaffolds thus prepared comprising
hybrid fibers of chitosan and acidic biopolymers and the scaffolds
to which cartilage tissue is adhered can be used as a graft for
repairing cartilage damage.
[0076] Culture of fibroblast is at least continued till
extracellular matrixes are formed. Usually by 2 to 4 weeks,
fibroblast is adhered on the surface of the three dimensional
scaffolds of the present invention, proliferate and secret
extracellular collagen-like matrix. Culture may be optionally
effected for about 2 months to prepare sufficient tissue for
implantation in vitro.
[0077] The three dimensional scaffolds thus prepared comprising
hybrid fibers of chitosan and acidic biopolymers and the scaffolds
to which fibroblast is adhered can be suitably used as a graft for
repair of ligament and tendon damage.
[0078] In the case of undifferentiated cells, for example,
mesenchyme stem cells are isolated from bone marrow liquid by a
density-gradient centrifugation. It is possible to add growth
factors such as TGF-beta and FGF to a medium such as DMEM to
differentiate the stem cell to chondrocyte or fibroblast before
seeding the cells on the three dimensional scaffolds, proliferating
and differentiating them. Alternatively undifferentiated cells may
be seeded on the scaffolds.
[0079] It is possible to add EGF (epidermal growth factor) to a
medium using nervous stem cell to differentiate it to nervous cell.
Alternatively, seeding of the cells on the scaffolds may be
effected in undifferentiated cells.
[0080] The prestnt invention will be illustrated by referring to
examples. However, the present invention is never limited to the
examples
EXAMPLES
Example 1
Preparation of Chitosan and Hyaluronic Acid Hybrid Fiber (1) and
(2)
[0081] A solution in which 8 (w/v)% chitosan (Kimitu Chemical
Industry, F2P, Molecular weight: about 165,000) was dissolved in 4%
aqueous acetic acid solution was packed with a column (glass, 45 mm
in inner diameter, 45 mm in length) and filtered through a
filtering cloth under compression (0.6 kgf/cm.sup.2). The filtrate
was packed with a spinning column (glass, inner diameter 45 mm,
length 410 mm) and used as a spinning liquid to prepare fibers with
a simple spinning apparatus in the following method: The spinning
solution above was extruded through a nozzle having 50 holes (.phi.
0.1 mm) under a compression of 0.8 kgf/cm.sup.2 into saturated
calcium chloride aqueous solution (the first coagulating bath:
water/methanol=1/1 (by volume); 100 cm in bath length; about 2 L in
volume) to form fibers. The fibers were then immersed in
water/methanol=1/1 by volume (the second coagulating bath: 50 cm in
bath length, about 1 L in volume), further immersed in 0.05%
hyaluronic acid solution (water/methanol=1/1 (by volume)) (the
third bath: 50 cm in bath length, about 150 mL in volume). The
fibers were then passed through rollers (first roller, speed 3.2
m/min; second roller, speed 3.2 m/min; draw ratio 1.0) and winded
up with a wind-up roller. The fibers were immersed in 0.8 (w/v)%
sodium hydroxide solution (water/methanol=1/9 (by volume)) for 15
hours, then washed with water, further immersed in methanol for
about 2 hours and air dried at room temperature, or removed from
the roller and air dried at a room temperature to give soft and
tender chitosan-hyaluronic acid hybrid fibers (referred to as
"chitosan-hyaluronic acid hybrid fiber (1)" hereinafter)
[0082] Another fibers were prepareed in the similar method except
that the concentration of hyarulonic acid is 0.1 (w/v) % to give a
soft and tender chitosan-hyaluronic acid hybrid fibers (referred to
as "chitosan-hyaluronic acid hybrid fiber (2)" hereinafter).
Comparative Example 1
Preparation of Hybrid Fiber without After-Treatment
[0083] Fibers were prepared in the same condition as
chitosan-hyaluronic acid hybrid fiber (1) except that the
after-treatment with sodium hydroxide solution after the spinning
was not effected.
Comparative Example 2
Preparation of Chitosan (Alone) Fiber (a)
[0084] Chitosan (alone) fibers were prepared in the similar way as
Example 1 except that hyaluronic acid is not added in the third
bath (referred to as "chitosan fiber (a)" hereinafter).
Example 2
Preparation of Chitosan and Hyaluronic Acid Hybrid Fiber (3) and
(4)
[0085] A solution in which 3.5 (w/v)% chitosan (Kimitu Chemical
Industry, B, Molecular weight: about 600,000) was dissolved in 2%
acetic acid aqueous solution was packed with a column and filtered
through a filtering cloth under compression. The filtrate was
packed with a spinning column and used as spinning liquid to
prepare fibers using a simple spinning apparatus in the following
method: The spinning solution above was extruded through a nozzle
having 50 holes (.phi. 0.1 mm) under a compression of 0.8
kgf/cm.sup.2 into 53 (w/v) % calcium chloride solution (the first
coagulating bath: water/methanol=1/1 (by volume), 100 cm in bath
length, about 2 L in volume) to form fibers. The fibers were then
immersed in water/methanol=1/1 by volume (the second coagulating
bath: 50 cm in bath length, about 1 L in volume), further immersed
in 0.05% hyaluronic acid solution (water/methanol=1/1 (by volume))
(the third bath: 50 cm in bath length, about 150 mL in volume). The
fibers were then passed through rollers (first roller, speed 4.4
m/min; second roller, speed 4.5 m/min; draw ratio 1.02) and winded
up with a wind-up roller. The fibers were immersed in 0.2% sodium
hydroxide solution (water/methanol=1/9 (by volume)) for 15 hours,
then washed with water, further immersed in methanol for about 2
hours and air dried at room temperature, or removed from the roller
and air dried at room temperature to give soft and tender
chitosan-hyaluronic acid hybrid fibers (referred to as
"chitosan-hyaluronic acid hybrid fiber (3)" hereinafter)
[0086] Another fibers were prepared in the similar method except
that the concentration of hyarulonic acid is 0.1% to give soft and
tender chitosan-hyaluronic acid hybrid fibers (referred to as
"chitosan-hyaluronic acid hybrid fiber (4)" hereinafter).
Comparative Example 3
Preparation of Chitosan (Alone) Fiber (b)
[0087] Chitosan (alone) fibers were prepared in the similar way as
Example 2 except that hyaluronic acid is not added in the third
bath (referred to as "chitosan fiber (b)" hereinafter).
Example 3
Preparation of Hybrid Fibers Using Various After-Treating
Agents
[0088] Chitosan-hyarulonic acid hybrid fibers were prepared in the
similar methods as in Example 2 (hyarulonic acid concentration
0.05%) except that the following after-treating agents are used in
place of 0.2% (w/v) sodium hydroxide solution (water/methanol=1/9
(by volume)). TABLE-US-00001 TABLE 1 Aftertreating agent Concn.
Solvent composition Potassium Carbonate 2% water/ (volume) methanol
= 1/9 Sodium Carbonate 2% water/ '' methanol = 1/1 Sodium
bicarbonate 1% water/ '' methanol = 1/1 Sodium bicarbonate 3%
water/ '' methanol = 3/2 Tripotassium Phosphate 2% water/ ''
methanol = 1/1 Dipotassium hydrogen 2% water/ '' phospate methanol
= 1/1 Sodium dihydrogen 2% water/ '' phospate methanol = 1/1 Sodium
sulfate 2% water/ '' methanol = 3/2 Citric Acid 3% water/ ''
methanol = 1/9
Example 4
Preparation of Hybrid Fiber Using Various Coagulants
[0089] A solution in which 3.5 (w/v)% chitosan (Kimitu Chemical
Industry, B, Molecular weight: about 600,000) was dissolved in 2%
acetic acid aqueous solution) was packed with a column and filtered
through a filtering cloth under compression. The filtrate was
packed with a spinning column and used as a spinning liquid to
prepare fibers using a simple spinning apparatus in the following
method: The spinning solution above was extruded through a nozzle
having 50 holes (.phi. 0.1 mm) under a compression of 0.8
kgf/cm.sup.2 into the various coagulating liquids shown in Table 2
(first coagulating bath: 100 cm in bath length, about 2 L in
volume) to form fibers. The fibers were then immersed in
water/methanol=1/1 by volume (second coagulating bath: 50 cm in
bath length, about 1 L in volume), further immersed in 0.05%
hyaluronic acid solution (water/methanol=1/1 (by volume)) (50 cm in
bath length, about 150 mL in volume). The fibers were passed
through rollers (first roller, speed 4.4 m/min; second roller,
speed 4.5 m/min; draw ratio 1.02) and winded up with a wind-up
roller. The fibers were immersed in water for 10 minutes, washed
again with water, further immersed in methanol for about 2 hours,
followed by drying to give fibers. TABLE-US-00002 TABLE 2 Coagulant
Concn. Solvent composition Sodium hydroxide 5% water/methanol = 1/1
(volume) Sodium carbonate 5% water/methanol = 1/1 (volume)
Tripotasium phosphaate 5% water/methanol = 1/1 (volume) Sodium
sulfate 5% water/methanol = 1/1 (volume)
Example 5
Preparation of Chitosan and Alginic Acid Hybrid Fiber
[0090] Chitosan and alginic acid hybrid fibers were prepared in the
similar way as in Example 2 except that 0.05% and 0.1% alginic acid
were used respectively in place of 0.05% hyaluronic acid
Example 6
Tensile Strength of Chitosan and Hyaluronic Acid Hybrid Fiber
[0091] Tensile strength and elongation of each fiber prepared in
Examples 1 and 2 were measured. The load at break and elongation
were measured in accordance with JIS standard L1015. The sectional
area of each fibers were measured by image processing under a
microscope.
Method for Measuring Breaking Force and Strain:
[0092] Each fiber in the form of thread (prepared by binding 50
monofilaments) was cut to about 40 mm in length, both the ends
thereof were put in the adhesive paper (for example, using post-it)
to prepare each sample in which the distance between gages is 20
mm. The both ends of the sample are fixed to clip-type gripping
tools (product number 343-06742-03, Shimadzu Corporation, Japan)
and the upper gripping tool is suspended to a load cell (20 N,
product number 346-51294-03, Shimadzu Corporation, Japan) before it
was set to a desk-type material testing machine (AGS-H, product
number 346-51299-02, Shimadzu Corporation, Japan). It was
vertically pulled in a pulling rate of 20 mm/min and breaking force
and strain were measured from the force and displacement at
failure. These data are incorporated in a personal conputer (IBM,
NetVista A 40). A software (TRAPEZIUM, Shimadzu Corporation, Japan)
was used for measurement and analysis of data.
Method for Measuring Sectional Area of Fiber:
[0093] Each fiber in the form of thread (prepared by binding 50
monofilaments) was cut to about 5 mm in length, inserted into holes
in a plastic plate having small apertures of diameter of about 1 mm
and fixed to it. The plastic plate was placed on the dais of an
optical microscope (BX50, Olympus Optical Industry). The image of
fiber section was caught through a camera control unit (ICD-740,
Ikegami Tusinki) containing a CCD camera. The sectional area of the
fibers were determined by an image processing instrument (VIDEO
MICRO METER, Model VM-30, Olympus Optical Industry, Monitor Screen
TM150, Ikegami Tusinki). The strength and strain at failure of each
fiber are shown in Table 3. TABLE-US-00003 TABLE 3 Strength and
Strain of chitosan and hyarulonic acid hybrid fiber (1) and (2)
(Averaged value .+-. standard error, n = 5) Maximum Sectional
Strength Strain at breaking Area failure Fiber force (N)
(.mu.m.sup.2) (N/mm.sup.2) (%) Chitosan 1.56 .+-. 0.06 12199.64
.+-. 128.07 .+-. 4.26 .+-. Fiber (a) 205.47 4.87 0.41 Chitosan-
2.35 .+-. 0.08 15397.20 .+-. 152.61 .+-. 6.11 .+-. hyarulonic
187.03 5.39 0.72 acid hybrid fiber (1) Chitosan- 4.10 .+-. 0.16
18843.20 .+-. 217.55 .+-. 3.14 .+-. hyarulonic 284.69 8.47 0.28
acid hybrid fiber (2)
[0094] Although the strength of chitosan (alone) fiber is about 130
N/mm.sup.2, hybridization with hyaluronic acid increases it to
about 150-220 N/mm.sup.2. A fiber in which collagen fiber is
crosslinked with nordihydroguayaletinic acid has a strength of 50
N/mm.sup.2 (Koob, T. J., Et al., J. Biomed. Mater Res., 56:40-48
(2001). Accordingly, it was confirmed that chitosan-hyarulonic acid
hybrid fiber had about three- to five-fold strength compared with
collagen fiber TABLE-US-00004 TABLE 4 Strength and Strain of
chitosan and hyarulonic acid hybrid fiber (3) and (4) (Averaged
value .+-. standard error, n = 5) Maximum Sectional Strength Strain
at breaking Area failure Fiber force (N) (.mu.m.sup.2) (N/mm.sup.2)
(%) Chitosan 2.86 .+-. 0.03 15576.08 .+-. 183.60 .+-. 2.54 .+-.
Fiber (b) 332.20 1.65 0.16 Chitosan- 4.17 .+-. 0.08 19165.34 .+-.
217.60 .+-. 5.89 .+-. hyarulonic 133.35 3.93 0.26 acid hybrid fiber
(3) Chitosan- 3.58 .+-. 0.06 18031.82 .+-. 198.51 .+-. 4.55 .+-.
hyarulonic 243.88 3.47 0.22 acid hybrid fiber (4)
Example 7
Adhesion of Chondrocyte on Chitosan and Hyarulonic Acid Hybrid
Fiber
[0095] In order for chondrocyte to proliferate and differentiate on
a scaffold, it is necessary for chondrocytes to adhere to a
scaffold as much as possible. The adhesion of chitosan (alone)
fiber and chitosan-hyaluronic acid hybrid fiber prepared in Example
2 to chondrocytes was determined. Commercially available degradable
seaming thread for medical use (biodegradable synthetic polymer,
polyglactin-910, Vicryl3-0, Ethicon Co., NJ, USA) was used as a
control.
[0096] Chondrocytes were isolated from the articular surfaces of a
Japanese white rabbit (8 weeks age, body weight 1.8-2.0 kg). The
concentration of the cell was 2.times.10.sup.6 cells/ml. The
evaluation of adhesion was in accordance with a method of Nishimura
(NIshimura, J. Biol. Macromol, 7:100-104, 1985). That is, each
fibers were cut to 10 mm in length and a fixed amount of the fiber
(100 mg) was packed into a teflon tube (5 mm in inner diameter, 30
mm in length). One hundred microliter of the suspension containing
chondrocytes was loaded onto one end of the tube and incubated at
37.degree. C. for 1 hour. One mililitter of PBS (phosphate buffered
saline) was then passed through the tube to give an eluted
solution. The number of cells in the effluent was counted and the
ratio of cell effluence was calculated. TABLE-US-00005 TABLE 5
Comparison of chondrocyte adhesion to various fibers Percent
effluence of cell Sample (average .+-. standard error, n = 5)
Vicryl 64.2 .+-. 7.5 chitosan fiber 13.0 .+-. 2.5*
chitosan-hyarulonic acid 11.1 .+-. 6.4* fiber (3)
chitosan-hyarulonic acid 26.9 .+-. 6.6* fiber (4) *p < 0.05 vs
Vicryl
[0097] As shown above, cell adhesion between Vicryl and the fibers
of the present invention is significantly different in ANOVA
statistical analysis.
Example 8
Adhesion of Fibroblast on Chitosan and Hyarulonic Acid Hybrid
Fiber
[0098] In order for fibroblast to proliferate and differentiate on
a scaffold, it is necessary for fibroblast to adhere to the three
dimensional scaffold as much as possible. The adhesion of chitosan
(alone) fiber and chitosan-hyaluronic acid hybrid fiber prepared in
Example 1 to fibroblast was determined. Commercially available
degradable seaming thread for medical use (biodegradable synthetic
polymer, polyglactin-910, Vicryl, Ethicon Co., NJ, USA) was used as
control.
Test Method:
[0099] Fibroblasts were isolated from the pateller tendon of a
Japanese white rabbit (8 weeks age, body weight 1.8-2.0 kg). The
concentration of the fibroblast was 1.times.10.sup.7 cells/ml. The
evaluation of the adhesion was in accordance with the method of
Nishimura (Nishimura, J. Biol. Macromole, 7:100-104, 1985). That
is, each fibers were cut to 10 mm in length and a fixed amount of
the fibers were packed into a Teflon (Trade Mark) tube (5 mm in
inner diameter, 30 mm in length). One milliliter of the solution
containing fibroblasts was loaded onto one end of the tube and
incubated at room temperature for 15 minutes. One milliliter of PBS
(phosphate buffered saline) was then passed through the tube to
give an eluted solution. The number of cells in the effluent was
counted and is considered to be the number of cell which did not
adhere to the fiber. TABLE-US-00006 TABLE 6 Comparison of
fibroblast adhesion to various fibers number of effluent fibroblast
Sample (average .+-. standard error, n = 5) Vicryl 151.1 .+-. 4.8
Chitosan fiber 44.4 .+-. 7.2* Chitosan-hyarulonic acid 12.0 .+-.
3.1*,** fiber (1) Chitosan-hyarulonic acid 14.8 .+-. 7.7*,** fiber
(2) *p < 0.05 vs Vicryl **p < 0.05 vs chitosan fiber
[0100] As shown above, there is significant difference in ANOVA
statistical analysis in cell adhesion between Vicryl and the fibers
of biopolymers. Statistically significant difference is observed
between chitosan (alone) fiber and the chitosan-hyarulonic acid
hybrid fiber, demonstrating that the chitosan-hyarulonic acid
hybrid fiber has better adhesion to fibroblast
Example 9
Shape Stability of Fiber in Medium
[0101] About 20 mg of each of the chitisan-hyaluronic hybrid fiber
prepared in Examples 1-4, the chitisan-hyaluronic hybrid fiber
prepared in Comparative Examples 1, the chitosan (alone) fiber
prepared in Comparative Examples 2 and 3 was placed in a tube,
respectively, and 2 ml of DMEM (Dulbecco's Modified Eagle's Medium,
Sigma, Code D5796) supplemented with 10% FBS (fetal bovine serum)
was added to it and was left at room temperature for two weeks.
[0102] The hybrid fiber prepared in Comparative Example 1 in which
calcium chloride was used as coagulating agent and the
after-treatment was not effected became unclean in shape and the
color of medium was changed to yellow. In other fibers, shape
change was not observed and the color of medium remains red.
Example 10
Preparation of Three Dimensional Scaffolds from Chitosan and
Hyaluronic Acid Hybrid Long Fiber
[0103] Long fibers of more than 100 m in length was prepared from
the fibers in which spinning were effected in the method described
in Example 1 followed by the treatment sodium hydroxide and
methanol. After the long fibers are subjected to twisting to form
twisted yarn, a zonary structure was prepared from the yarn using
commercially available braider. The three dimensional scaffolds
having a fixed shape were prepared using it.
Example 11
Culture of Chondrocyte Using Three Dimensional Scaffold
[0104] Culture test was effected using the three dimensional
scaffolds prepared in Example 10. Chondrocytes were collected and
cultured in accordance with methods of Kawasaki (Kawasaki, K., et
al., J. Cell Physiol., 179:142-148 (1999) and Yasui (Yasui, N., et
al., Exp. Cell Biol., 50:92-100(1982)). That is, a piece of
cartilage tissue was isolated from the articular surfaces of a
Japanese white rabbit (8 weeks age, body weight 1.8-2.0 kg) and
treated at 37.degree. C. for 25 minutes by adding 0.25% trypsin
solution before treating at 37.degree. C. for 25 minutes by adding
0.25% collagenase (Type II) solution to isolate cells. After 50
.mu.l of this cell suspension was removed, 50 .mu.l of Trypan blue
was added, the suspension was thoroughly stirred. Twenty
microliters of the suspension were placed on hemocytometer and the
number of the cells was counted to calculate the total number of
the cells.
[0105] The three dimensional scaffolds of the chitosan and
hyarulonic hybrid fibers which have been previously sterilizd in an
autoclave were placed in a multi-well plate (12 wells, Falcon). One
hundred microlitter of a suspention containing chondrocytes were
added on each scaffold in well so that each well contained
5.times.10.sup.5 chondrocytes cells. After incubating under 5%
CO.sub.2 in an incubator at 37.degree. C. for 1 hour, 2 ml of DMEM
medium was added by portion and 20 .mu.l of 0.1% ascorbic acid
phosphate was further added and the mixture was incubated under the
above conditions.
[0106] FIG. 1 shows an optical micrograph of chondrocytes after 21
days of culture. It is confirmed that seeded chondrocytes
proliferate well on the fiber and in aperture between the fibers.
FIG. 2 shows the result of Arcyanblue Safranin staining which
confirm many extracellular matrixes stained strongly in blue. It is
known that the chondrocytes are favorably proliferate and
differentiate on the fiber and secret extracellular matrixes such
as chondroitin sulfate. FIG. 3 shows an produced amount of proteins
and acid mucopolysaccharides per the three dimensional scaffold
after 1, 7, 14 and 21 days of culture. The amount of produced
proteins and acid mucopolysaccharides increased with culture. From
these facts, it is known that good proliferation and
differentiation of chondrocyte produce various proteins and
extracellular matrixes such as chondroitin sulfate.
Example 12
Evaluation of Cartilage Tissue Regeneration in Implantation of
Cultured Three Dimensional Scaffold into Animal
[0107] A defective portion of about 4.times.6.times.1.5 mm on
articular cartilage tissue of the knee was prepared under
anesthesia in Japanese white rabbit (8 weeks age, body weight
1.8-2.0 kg). To this place, the three dimensional scaffold of
Example 10 on which chondrocytes of rabbit had been cultured for
two weeks was implanted and both the sides of the scaffold were
lightly fixed with a seaming thread. On 8 weeks after the
implantation, the defective portion was subjected to laparotomy and
tissue observation was effected with Safranin O staining. Cartilage
tissue which was stained in red was observed in the defective
portion to which the three dimensional scaffold was implanted,
confirming that cartilage tissue was regenerated. Fixation of the
scaffold to the cell was also good and it was observed that the
scaffold has been gradually decomposed. Extravastion of
inflammatory cell associated with the scaffold implantation was
scarcely observed. Accordingly, it is considered that no strong
immune response associated with the scaffold implantation
occurred.
Example 13
Culture of Fibroblast Using Three Dimensional Scaffold
[0108] Fibroblasts were collected and cultured in accordance with
Matin's method (Martin, G. M., Tissue Culture, Methods and
Application, Academic Press, 39, 1973). A piece of 2 mm.times.2 mm
was prepared from the patellar tendon of a Japanese white rabbit (8
weeks age, body weight 1.8-2.0 kg) and covered with a cover glass
and fixed to a dish having a diameter of 35 mm. To this, DMEM
supplemented with 10% FBS was added and the mixture was incubated
under 5% CO.sub.2 in a incubator at 37.degree. C. for two weeks.
When fibroblasts became confluent, medium was removed and
fibroblasts were washed with PBS (-). 0.5 ml of 0.25% trypsin was
added and incubation was effected at 37.degree. C. for 15 minutes,
followed by the addition of 1 ml of the medium to obtain cell
suspension. To this suspension, 50 .mu.l of 0.04% Trypan Blue were
added and the number of the cells was counted using a
hemocytometer.
[0109] The three dimensional scaffolds of chitosan and hyarulonic
hybrid fiber which had been previously sterilized in an autoclave
were placed in a plate (12 holes). One hundred microliters of
solution containing fibroblasts were added on each scaffold in well
so that each well contained 1.times.10.sup.5 cells. After
incubating under 5% CO.sub.2 in an incubator at 37.degree. C. for 1
hour, 2 ml of DMEM medium was added and the mixture was incubated
under the above conditions. The amount of DNA was determined after
1, 7, 14 and 21 days of culture as an index of cell number.
[0110] The measurement was effected in accordance with Rago's
method (Rago, R. et al., Anal. Biochem., 191:31-34 (1990)). That
is, sodium chloride was added to 0.05 M phosphate buffer (pH 7.4)
to make 2M solution of sodium chloride. Cultured fibroblasts were
removed together with the scaffold and the medium was washed with
PBS. The scaffolds are finely cut with scissors before transferring
it into a 1.5 ml tube. One mililiter of 0.05 M phosphate buffer
(containing 2 M sodium chloride, pH 7.4) was added to the tube,
stirred thoroughly and left at room temperature for 1 minute. After
the solution was enough stirred again, the supernatant thereof was
used as a sample solution. To 100 .mu.l of the sample solution, 2
ml of 0.05 M phosphate buffer (containing 2 M sodium chloride, pH
7.4) was added, followed by the addition of 10 .mu.l of a
fluorescent reagent (Hoechst 33258, 0.02% solution was prepared
using purified water). After the solution was thoroughly stirred,
the intensity of fluorescence of the resulting solution was
measured with a spectrofluorometer (RT-5300PC, Shimadzu
Corporation, Japan)(excitation at 356 nm; emission at 458 nm). A
calibration curve was made using standard DNA solution in the range
of 0-125 .mu.g/mL. The DNA concentration contained in the sample
solution was determined from the calibration curve. As shown in
FIG. 5, the amount of DNA generated increase with culturing,
confirming that fibroblasts benignly proliferate on the three
dimensional scaffold.
[0111] FIG. 6 shows the result of immunohistochemicalstaining of
type I collagen (cultured on the three dimensional scaffold for 28
days) by the streptavidin-biotin method. Eextracellular substrate
was stained and it was demonstrated that the three dimensional
scaffold prepared from the hybrid fibers of the present invention
was excellent in the production of type I collagen which is an
extracellular matrix of ligament and tendon.
[0112] FIG. 7 show the image by a scanning electron microscopy
obtained when culturing was effected for 28 days using the three
dimensional scaffold. Samples were prepared as follows: 0.1 M
phosphate buffer (pH7.2) was prepared. To 200 ml of the phosphate
buffer, 6.85 g of sucrose was added to prepare 0.1 M phosphate
buffer containing 0.1 M sucrose. Likewise, 0.2 M phosphate buffer
(pH7.2) containing 0.2 M sucrose was also prepared. In addition, 2%
osmic acid solution was diluted twice with 0.2 M phosphate buffer
(pH7.2) containing 0.2 M sucrose to prepare 1% osmic acid
solution.
[0113] The scaffolds cultured for 28 days were immersed in 0.1 M
phosphate buffer (pH7.2) containing 0.1 M sucrose, and left at
37.degree. C. for 10 minutes. The procedure was repeated three
times before the scaffolds were immersed in 2% glutaraldehyde
solution (prepared with 0.1 M phosphate buffer), left at 37.degree.
C. for 1 hour and washed. The scaffolds containing the cells was
immersed in 0.1 M phosphate buffer containing 0.1 M sucrose, left
at 37.degree. C. for 1 hour, and washed. The procedure was repeated
three times before the scaffolds were immersed in 1% osmic acid
solution at room temperature to be subjected to after-fixation
treatment. Further in order to effect conductivity staining, the
scaffolds were washed with 0.1 M phosphate buffer before they were
immersed in 2% aqueous tannic acid solution for two hours followed
by the immersion in 0.1 M phosphate buffer for two hours. The
scaffolds were then immersed in 2% osmic acid containing 0.34 g/10
mL of sucrose for two hours followed by the immersion in 0.1 M
phosphate buffer for 2 hours.
[0114] The scaffolds were immersed for 10 minutes in aqueous
ethanol solutions in an order (20-99.5% ethanol) in which the
concentration of ethanol was increased at 10% interval. It was
finally immersed in absolute ethanol twice to be dehydrated. The
scaffolds were then immersed twice in isoamyl acetate for 30
minutes to change solvent from ethanol to isoamyl acetate. Each
sample which was subjected to a critical point drying treatment
(temperature: 31.degree. C., pressure: 72.8 atm.) using liquidized
CO.sub.2 as a shifting solution. Each sample was then subjected to
a platinum-palladium vacuum evaporation treatment with an
ion-spattering equipment (E-102, Hitachi Ltd.) and then the state
of proliferation and differentiation of fibroblasts on the
scaffolds was observed. As results, fibroblasts proliferated and
differentiated on the surface of the three dimensional scaffolds
and much fibrous substance which were considered to be collagen was
observed (FIG. 7).
* * * * *